Publications by authors named "David K Chalmers"

60 Publications

Interaction with biliary and pancreatic fluids drives supersaturation and drug absorption from lipid-based formulations of low (saquinavir) and high (fenofibrate) permeability poorly soluble drugs.

J Control Release 2021 Mar 12;331:45-61. Epub 2021 Jan 12.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde Parkville, Victoria 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde Parkville, Victoria 3052, Australia. Electronic address:

Drug absorption from lipid-based formulations (LBFs) in the gastrointestinal (GI) tract is the result of a series of processes, including formulation dispersion, interaction with biliary and pancreatic secretions, drug solubilisation and supersaturation, and finally intestinal permeability. Optimal formulation design is dependent on a good understanding of the limitations to, and drivers of, absorption, but for LBFs the complexity of these processes makes data interpretation complex. The current study has re-examined a previous in vitro digestion-in situ perfusion model to increase physiological relevance and has used this model to examine drug absorption from LBFs. The composition of rat bile and jejunal fluid was also characterised to identify in vivo-relevant conditions. Digestion was initiated using rat bile/pancreatic fluid and the formulation and digestive enzymes mixed immediately prior to entry into the jejunum (allowing dilution/digestion to occur at the absorptive site). These conditions were employed to study drug absorption from LBFs of high (fenofibrate, FFB) and low (saquinavir, SQV) permeability compounds. The impact of polymeric precipitation inhibitors (PPIs) was also evaluated. For FFB, supersaturation, initiated by formulation interaction with biliary/pancreatic fluids, appeared to drive absorption and the addition of the PPIs poly(glycidyl methacrylate) (PPGAE) and hydroxypropylmethyl cellulose (HPMC), reduced drug precipitation, increased FFB supersaturation and increased absorption from a Type IV LBF of FFB. For a Type IIIB LBF however, PPIs were ineffective at increasing absorption. The impact of PPIs on the absorption of a less permeable drug, SQV, was similarly evaluated and again drug absorption appeared to be related to the extent of supersaturation, although in this case PPI were unable to promote absorption. For both FFB and SQV, drug absorption patterns obtained with the in vitro digestion-in situ perfusion mode, correlated well with in vitro supersaturation data and in vivo drug exposure data from oral bioavailability studies. The data are consistent with a mode of drug absorption where rapid dilution of LBFs with biliary and pancreatic secretions at the absorptive site in the upper small intestine drives transient supersaturation, that supersaturation is a significant driver of drug absorption for both low and high permeability drugs, and that PPIs delay drug precipitation, enhance supersaturation and promote drug absorption in a drug and formulation specific manner.
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http://dx.doi.org/10.1016/j.jconrel.2021.01.007DOI Listing
March 2021

Aqueous phase behavior of the PEO-containing non-ionic surfactant CE: A molecular dynamics simulation study.

J Colloid Interface Sci 2021 Apr 17;588:257-268. Epub 2020 Dec 17.

Medicinal Chemistry Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

Hypothesis: Non-ionic surfactants containing polyethylene oxide (PEO) chains are widely used in drug formulations, cosmetics, paints, textiles and detergents. High quality molecular dynamics models for PEO surfactants can give us detailed, atomic-scale information about the behavior of surfactant/water mixtures.

Simulations: We used two molecular dynamics force fields (FFs), 2016H66 and 53A6, to model the simple non-ionic PEO surfactant, hexaoxyethylene dodecyl ether (CE). We investigated surfactant/water mixtures that span the phase diagram of starting from randomly distributed arrangements. In some cases, we also started with prebuilt, approximate models. The simulations results were compared with the experimentally observed phase behavior.

Findings: Overall, this study shows that the spontaneous self-assembly of PEO non-ionic surfactants into different colloidal structures can be accurately modeled with MD simulations using the 2016H66 FF although transitions to well-formed hexagonal phase are slow. Of the two FFs investigated, the 2016H66 FF better reproduces the experimental phase behavior across all regions of the CEwater phase diagram.
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http://dx.doi.org/10.1016/j.jcis.2020.12.032DOI Listing
April 2021

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1.

ACS Pharmacol Transl Sci 2020 Aug 29;3(4):690-705. Epub 2020 Apr 29.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3010, Australia.

Cell-cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS). While several experimental structures show how NT binds NTS, details about the structural dynamics of NT during and after binding NTS, or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS, residue Y11 can exist in two χ rotameric states, gauche plus (g) or gauche minus (g). Since only the g Y11 state is observed in all the structures solved to date, we asked if the g state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS with the same affinity as NT10-13 but did not activate NTS, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS.
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http://dx.doi.org/10.1021/acsptsci.0c00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432660PMC
August 2020

Improving Membrane Permeation in the Beyond Rule-of-Five Space by Using Prodrugs to Mask Hydrogen Bond Donors.

ACS Chem Biol 2020 08 20;15(8):2070-2078. Epub 2020 Jul 20.

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

A wide range of drug targets can be effectively modulated by peptides and macrocycles. Unfortunately, the size and polarity of these compounds prevents them from crossing the cell membrane to reach target sites in the cell cytosol. As such, these compounds do not conform to standard measures of drug-likeness and exist in beyond the rule-of-five space. In this work, we investigate whether prodrug moieties that mask hydrogen bond donors can be applied in the beyond rule-of-five domain to improve the permeation of macrocyclic compounds. Using a cyclic peptide model, we show that masking hydrogen bond donors in the natural polar amino acid residues (His, Ser, Gln, Asn, Glu, Asp, Lys, and Arg) imparts membrane permeability to the otherwise impermeable parent molecules, even though the addition of the masking group increases the overall compound molecular weight and the number of hydrogen bond acceptors. We demonstrate this strategy in PAMPA and Caco2 membrane permeability assays and show that masking with groups that reduce the number of hydrogen-bond donors at the cost of additional mass and hydrogen bond acceptors, a donor-acceptor swap, is effective.
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http://dx.doi.org/10.1021/acschembio.0c00218DOI Listing
August 2020

Markov State Model Analysis of Haloperidol Binding to the D Dopamine Receptor.

J Chem Theory Comput 2020 Jun 6;16(6):3879-3888. Epub 2020 May 6.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde, Parkville, Victoria 3052, Australia.

We have developed Markov state models (MSMs) and hidden Markov models (HMMs) that describe the binding of haloperidol to the D dopamine receptor. Haloperidol is an antipsychotic drug that binds with nanomolar affinity to the D dopamine receptor, where it functions as an inverse agonist. The models were constructed using an adaptive sampling approach from 519 individual molecular dynamics simulations totaling 122 μs of simulated time and encompass the entire drug binding process. They reveal short-lived metastable bound states and two distinct long-lived bound conformations that cannot be separated in affinity using our current methodology. This work extends the use of MSMs and HMMs to study ligand binding, which thus far has been limited to simpler systems.
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http://dx.doi.org/10.1021/acs.jctc.0c00013DOI Listing
June 2020

INPHARMA-Based Determination of Ligand Binding Modes at α -Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity.

Chemistry 2020 Sep 18;26(51):11796-11805. Epub 2020 Aug 18.

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, 3010, VIC, Australia.

The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α -AR and α -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α -AR and α -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.
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http://dx.doi.org/10.1002/chem.202000642DOI Listing
September 2020

Production of metabolites of the anti-cancer drug noscapine using a P450 mutant library.

Biotechnol Rep (Amst) 2019 Dec 24;24:e00372. Epub 2019 Aug 24.

Department of Chemical Engineering, The University of Melbourne, Parkville, VIC, 3010, Australia.

Cytochrome P450 enzymes are a promising tool for the late-stage diversification of lead drug candidates and can provide an alternative route to structural modifications that are difficult to achieve with synthetic chemistry. In this study, a library of P450 mutants was produced using site-directed mutagenesis and the enzymes screened for metabolism of the opium poppy alkaloid noscapine, a drug with anticancer activity. Of the 18 enzyme mutants screened, 12 showed an ability to metabolise noscapine that was not present in the wild-type enzyme. Five noscapine metabolites were detected by LC-MS/MS, with the major metabolite for all mutants being demethylated noscapine. The highest observed regioselectivity for -demethylation was 88%. Two hydroxylated metabolites, a catechol and two - cleavage products were also detected. P450-mediated production of hydroxylated and -demethylated noscapine structures may be useful for the development of noscapine analogues with improved biological activity. The variation in substrate turnover, coupling efficiency and product distribution between the active mutants was considered alongside docking experiments to gain insight into structural and functional effects of the introduced mutations. Selected mutants were identified as targets for further mutagenesis to improve activity and when coupled with an optimised process may provide a route for the preparative-scale production of noscapine metabolites.
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http://dx.doi.org/10.1016/j.btre.2019.e00372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728265PMC
December 2019

Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa.

Toxicon 2019 Oct 11;168:104-112. Epub 2019 Jul 11.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, VIC, 3052, Australia. Electronic address:

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.
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http://dx.doi.org/10.1016/j.toxicon.2019.07.002DOI Listing
October 2019

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL.

J Pharm Sci 2019 Jan 28;108(1):193-204. Epub 2018 Nov 28.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052, Australia. Electronic address:

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6 and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development.
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http://dx.doi.org/10.1016/j.xphs.2018.11.028DOI Listing
January 2019

Improvement in the Predicted Partitioning of Alcohol and Polyethylene Oxide Groups Between Water and Octanol (logP) in Molecular Dynamics Simulations.

J Pharm Sci 2019 Jan 20;108(1):214-222. Epub 2018 Nov 20.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052, Australia.

Molecular dynamics simulations can be applied to explore the complex liquid phase behavior of lipid-based formulations and the gastrointestinal tract lumen. In order for the results from these simulations to be of value, the manner in which molecules interact with both aqueous and oil phases present needs to be as correct as possible. An existing molecular dynamics force field, GROMOS 53a6, was demonstrated to poorly reproduce the partitioning of straight-chain alcohol and short-chain polyethylene glycol (PEG) molecules between octanol and water phase (logP), with the molecules too hydrophobic. Force field parameters for Lennard-Jones interactions between CH2 and CH3 with water oxygen were adjusted to reproduce the experimental octanol logP, with all other Lennard-Jones and force field parameters left untouched. This parameter set, called 53a6, was subsequently used to recalculate straight-chain alcohol and short-chain PEG molecules, with significant improvement in the values obtained. Simulations of a nonionic surfactant in water, octaethylene glycol monocaprylate, were also performed to observe the aggregation behavior. 53a6 demonstrated significant improvements in water interactions with the PEG chains, well hydrating the PEG groups, and allowing the formation of micelles. Further improvements and evaluation of the improved parameter set are ongoing.
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http://dx.doi.org/10.1016/j.xphs.2018.11.016DOI Listing
January 2019

Controlled Construction of Cyclic d / l Peptide Nanorods.

Angew Chem Int Ed Engl 2019 01 5;58(2):596-601. Epub 2018 Dec 5.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

Cyclic d / l peptides (CPs) assemble spontaneously via backbone H-bonding to form extended nanostructures. These modular materials have great potential as versatile bionanomaterials. However, the useful development of CP nanomaterials requires practical methods to direct and control their assembly. In this work, we present novel, heterogeneous, covalently linked CP tetramers that achieve local control over the CP subunit order and composition through coupling of amino acid side-chains using copper-activated azide-alkyne cycloaddition and disulfide bond formation. Cryo-transmission electron microscopy revealed the formation of highly ordered, fibrous nanostructures, while NMR studies showed that these systems have strong intramolecular H-bonding in solution. The introduction of inter-CP tethers is expected to enable the development of complex nanomaterials with controllable chemical properties, facilitating the development of precisely functionalized or "decorated" peptide nanostructures.
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http://dx.doi.org/10.1002/anie.201811910DOI Listing
January 2019

Location of Solvated Probe Molecules Within Nonionic Surfactant Micelles Using Molecular Dynamics.

J Pharm Sci 2019 Jan 3;108(1):205-213. Epub 2018 Nov 3.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052, Australia.

An iconic textbook that pharmaceutical scientists encounter in undergraduate courses is "Martin's Physical Pharmacy and Pharmaceutical Sciences." Within the chapter on Colloids, a figure indicates the location of solubilization of molecules within spherical, nonionic surfactant micelles. The surfactant consists of polyethylene glycol (PEG) hydrophilic headgroups and alkane chains for the hydrophobic tail. The figure shows benzene and toluene within the alkane core, salicylic acid (2-hydroxybenzoic acid) at the interface between the core and PEG chains, and then para-hydroxybenzoic acid (4-hydroxybenzoic acid) located between the PEG chains. Molecular dynamics simulations of octaethylene glycol monododecyl ether micelles were performed with a series of probe molecules, including those within the Martin's figure, to determine their solubilization location. Relative placement of molecules within the micelle was correct; however, some specifics were different. In particular, benzene and toluene are excluded from the core, and 4-hydroxybenzoic acid prefers to maintain contact with the core. A series of molecules containing 6 carbon atoms were also studied to determine the effects of cyclization (moves out of core), polar functionalization (anchored to interface), and aromatization (excluded from central core). Molecular dynamics was found to be a useful tool for gaining insight into interactions important in solubilization of molecules.
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http://dx.doi.org/10.1016/j.xphs.2018.10.055DOI Listing
January 2019

A Cyclic Peptide Inhibitor of the iNOS-SPSB Protein-Protein Interaction as a Potential Anti-Infective Agent.

ACS Chem Biol 2018 10 18;13(10):2930-2938. Epub 2018 Sep 18.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences , Monash University , Parkville Victoria 3052 , Australia.

SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4 interact with inducible nitric oxide synthase (iNOS), causing the iNOS to be polyubiquitinated and targeted for degradation. Inhibition of this interaction increases iNOS levels, and consequently cellular nitric oxide (NO) concentrations, and has been proposed as a potential strategy for killing intracellular pathogens. We previously described two DINNN-containing cyclic peptides (CP1 and CP2) as potent inhibitors of the murine SPSB-iNOS interaction. In this study, we report the crystal structures of human SPSB4 bound to CP1 and CP2 and human SPSB2 bound to CP2. We then used these structures to design a new inhibitor in which an intramolecular hydrogen bond was replaced with a hydrocarbon linkage to form a smaller macrocycle while maintaining the bound geometry of CP2 observed in the crystal structures. This resulting pentapeptide SPSB-iNOS inhibitor (CP3) has a reduced macrocycle ring size, fewer nonbinding residues, and includes additional conformational constraints. CP3 has a greater affinity for SBSB2 ( K = 7 nM as determined by surface plasmon resonance) and strongly inhibits the SPSB2-iNOS interaction in macrophage cell lysates. We have also determined the crystal structure of CP3 in complex with human SPSB2, which reveals the structural basis for the increased potency of CP3 and validates the original design.
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http://dx.doi.org/10.1021/acschembio.8b00561DOI Listing
October 2018

The influence and manipulation of acid/base properties in drug discovery.

Drug Discov Today Technol 2018 Jul 26;27:41-47. Epub 2018 Apr 26.

Faculty of Pharmacy and Pharmaceutical Sciences, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

There is a growing awareness of the importance of acid/base properties in medicinal chemistry research. In many drug classes, ionisable groups are present that make critical interactions with the receptor and are essential for potency. Yet the presence of these groups may cause problems with oral bioavailability, pharmacokinetics, or toxicity. Manipulating pK values during drug development or applying pro-drug techniques are strategies that can overcome potential deficits in a variety of these areas. Knowledge of drug ionisation states coupled with a consideration of pH-specific cellular environments can be used advantageously to target chemoresistance. As modern drug research ventures into drug candidates that exceed the rule of 5, such exploration requires an understanding of drug acid/base properties and how these factors affect ADMET characteristics.
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http://dx.doi.org/10.1016/j.ddtec.2018.04.003DOI Listing
July 2018

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV-1 Integrase.

ChemMedChem 2018 08 6;13(15):1555-1565. Epub 2018 Jul 6.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

The p75 splice variant of lens epithelium-derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre-integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X-ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV-1 integrase core domain. Although the X-ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low-energy solution and bound conformations.
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http://dx.doi.org/10.1002/cmdc.201800129DOI Listing
August 2018

Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation and Enhance Drug Absorption from a Type IV Lipid-Based Formulation.

Mol Pharm 2018 06 4;15(6):2355-2371. Epub 2018 May 4.

The ability of lipid-based formulations (LBFs) to increase the solubilization, and prolong the supersaturation, of poorly water-soluble drugs (PWSDs) in the gastrointestinal (GI) fluids has generated significant interest in the past decade. One mechanism to enhance the utility of LBFs is to prolong supersaturation via the addition of polymers that inhibit drug precipitation (polymeric precipitation inhibitors or PPIs) to the formulation. In this work, we have evaluated the performance of a range of PPIs and have identified PPIs that are sufficiently soluble in LBF to allow the construction of single phase formulations. An in vitro model was first employed to assess drug (fenofibrate) solubilization and supersaturation on LBF dispersion and digestion. An in vitro-in situ model was subsequently employed to simultaneously evaluate the impact of PPI enhanced drug supersaturation on drug absorption in rats. The stabilizing effect of the polymers was polymer specific and most pronounced at higher drug loads. Polymers that were soluble in LBF allowed simple processing as single phase formulations, while formulations containing more hydrophilic polymers required polymer suspension in the formulation. The lipid-soluble polymers Eudragit (EU) RL100 and poly(propylene glycol) bis(2-aminopropyl ether) (PPGAE) and the water-soluble polymer hydroxypropylmethyl cellulose (HPMC) E4M were identified as the most effective PPIs in delaying fenofibrate precipitation in vitro. An in vitro model of lipid digestion was subsequently coupled directly to an in situ single pass intestinal perfusion assay to evaluate the influence of PPIs on fenofibrate absorption from LBFs in vivo. This coupled model allowed for real-time evaluation of the impact of supersaturation stabilization on absorptive drug flux and provided better discrimination between the different PPIs and formulations. In the presence of the in situ absorption sink, increased fenofibrate supersaturation resulted in increased drug exposure, and a good correlation was found between the degree of in vitro supersaturation and in vivo drug exposure. An improved in vitro-in vivo correlation was apparent when comparing the same formulation under different supersaturation conditions. These observations directly exemplify the potential utility of PPIs in promoting drug absorption from LBF, via stabilization of supersaturation, and further confirm that relatively brief periods of supersaturation may be sufficient to promote drug absorption, at least for highly permeable drugs such as fenofibrate.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00206DOI Listing
June 2018

Computational Models of the Intestinal Environment. 3. The Impact of Cholesterol Content and pH on Mixed Micelle Colloids.

Mol Pharm 2017 11 5;14(11):3684-3697. Epub 2017 Oct 5.

Capsugel Research & Development, Parc d'Innovation , Strasbourg, France.

In this study, we use molecular dynamics (MD) and experimental techniques (nephelometry and dynamic light scattering) to investigate the influence of cholesterol content and pH on the colloidal structures that form in the gastrointestinal (GI) tract upon lipid digestion. We demonstrate that the ionization state of the molecular species is a primary driver for the self-assembly of aggregates formed by model bile and therefore should be considered when performing in silico modeling of colloidal drug delivery systems. Additionally, the incorporation of physiological concentrations of cholesterol within the model systems does not affect size, number, shape, or dynamics of the aggregates to a significant degree. The MD data shows a reduction in aggregate size with increasing pH, a preference for glycodeoxycholate (GDX) to occupy the aggregate surface, and that the mixed micellar aggregates are oblate spheroids (disc-like). The results obtained assist in understanding the process by which pH and cholesterol influence self-assembly of mixed micelles within the GI tract. The MD approach provides a platform for investigation of interactions of drugs and formulation excipients with the endogenous contents of the GI tract.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00446DOI Listing
November 2017

How kanamycin A interacts with bacterial and mammalian mimetic membranes.

Biochim Biophys Acta Biomembr 2017 Nov 26;1859(11):2242-2252. Epub 2017 Aug 26.

School of Chemistry, Monash University, Wellington Rd, Clayton, VIC 3800, Australia. Electronic address:

Biological membranes are natural barriers to the transport of molecules and drugs within human bodies. Many antibacterial agents need to cross these membranes to reach their target and elicit specific effects. Kanamycin A belongs to the family of aminoglycoside antibiotics that target cellular RNA to inhibit bacterial and viral replication. Previous studies have shown that aminoglycosides bind to mammalian but disrupt bacterial membranes. In this study, molecular dynamics (MD) simulations and infrared (IR) spectroscopy were applied to investigate the initial, first key interactions of kanamycin A, as a representative aminoglycoside, with both bacterial and mammalian lipid bilayers at the molecular level. Computational studies revealed strong hydrogen bonding interactions between the hydroxyl and amino groups of the aminoglycoside with the ester carbonyl and phosphate groups of the lipids. IR spectroscopy provided experimental verification of the important role of the lipid's ester carbonyl, phosphate and hydroxyl groups for aminoglycoside binding. The bacterial membrane became disordered upon aminoglycoside addition, whereas the mammalian membrane became stiffer and more ordered. This indicates the bacterial membrane disruption observed by previous studies.
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http://dx.doi.org/10.1016/j.bbamem.2017.08.016DOI Listing
November 2017

Free Energy Methods in Drug Design: Prospects of "Alchemical Perturbation" in Medicinal Chemistry.

J Med Chem 2018 02 4;61(3):638-649. Epub 2017 Aug 4.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University , 381 Royal Parade, Parkville, Victoria 3052, Australia.

Underpinning all drug discovery projects is the interaction between a drug and its target, usually a protein. Thus, improved methods for predicting the magnitude of protein-ligand interactions have the potential to improve the efficiency of drug development. In this review, we describe the principles of free energy methods used for the calculation of protein-ligand binding free energies, the challenges associated with these methods, and recent advances developed to address these difficulties. We then present case studies from 2005 to 2017, each demonstrating that alchemical free energy methods can assist rational drug design projects. We conclude that alchemical methods are becoming a feasible reality in medicinal chemistry research due to improved computational resources and algorithms and that alchemical free energy predictions methods are close to becoming a mainstream tool for medicinal chemists.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00681DOI Listing
February 2018

Parallel and antiparallel cyclic d/l peptide nanotubes.

Chem Commun (Camb) 2017 Jun 5;53(49):6613-6616. Epub 2017 Jun 5.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Nanotubes made from H-bonded cyclic d/l peptide (CP) subunits have great potential for the construction of nanomaterials of wide chemical and structural diversity but, to date, difficulties in structural characterisation have restricted development of these materials. We present the first crystal structures of continuous CP nanotubes with antiparallel and parallel stacking arrangements, assembled separately from two peptides; cyclo[(Asp-d-Leu-Lys-d-Leu)] and cyclo[(Asp-d-Ala-Lys-d-Ala)].
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http://dx.doi.org/10.1039/c7cc00846eDOI Listing
June 2017

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue.

Toxicon 2017 Apr 17;129:113-122. Epub 2017 Feb 17.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia. Electronic address:

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.012DOI Listing
April 2017

Computational Models of the Gastrointestinal Environment. 1. The Effect of Digestion on the Phase Behavior of Intestinal Fluids.

Mol Pharm 2017 03 15;14(3):566-579. Epub 2017 Feb 15.

Capsugel Research and Development , Strasbourg, France.

Improved models of the gastrointestinal environment have great potential to assist the complex process of drug formulation. Molecular dynamics (MD) is a powerful method for investigating phase behavior at a molecular level. In this study we use multiple MD simulations to calculate phase diagrams for bile before and after digestion. In these computational models, undigested bile is represented by mixtures of palmitoyl-oleoylphosphatidylcholine (POPC), sodium glycodeoxycholate (GDX), and water. Digested bile is modeled using a 1:1 mixture of oleic acid and palmitoylphosphatidylcholine (lysophosphatidylcholine, LPC), GDX, and water. The computational phase diagrams of undigested and digested bile are compared, and we describe the typical intermolecular interactions that occur between phospholipids and bile salts. The diffusion coefficients measured from MD simulation are compared to experimental diffusion data measured by DOSY-NMR, where we observe good qualitative agreement. In an additional set of simulations, the effect of different ionization states of oleic acid on micelle formation is investigated.
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http://dx.doi.org/10.1021/acs.molpharmaceut.6b00888DOI Listing
March 2017

Computational Models of the Gastrointestinal Environment. 2. Phase Behavior and Drug Solubilization Capacity of a Type I Lipid-Based Drug Formulation after Digestion.

Mol Pharm 2017 03 15;14(3):580-592. Epub 2017 Feb 15.

Capsugel Research and Development , Strasbourg, France.

Lipid-based drug formulations can greatly enhance the bioavailability of poorly water-soluble drugs. Following the oral administration of formulations containing tri- or diglycerides, the digestive processes occurring within the gastrointestinal (GI) tract hydrolyze the glycerides to mixtures of free fatty acids and monoglycerides that are, in turn, solubilized by bile. The behavior of drugs within the resulting colloidal mixtures is currently not well characterized. This work presents matched in vitro experimental and molecular dynamics (MD) theoretical models of the GI microenvironment containing a digested triglyceride-based (Type I) drug formulation. Both the experimental and theoretical models consist of molecular species representing bile (glycodeoxycholic acid), digested triglyceride (1:2 glyceryl-1-monooleate and oleic acid), and water. We have characterized the phase behavior of the physical system using nephelometry, dynamic light scattering, and polarizing light microscopy and compared these measurements to phase behavior observed in multiple MD simulations. Using this model microenvironment, we have investigated the dissolution of the poorly water-soluble drug danazol experimentally using LC-MS and theoretically by MD simulation. The results show how the formulation lipids alter the environment of the GI tract and improve the solubility of danazol. The MD simulations successfully reproduce the experimental results showing the utility of MD in modeling the fate of drugs after digestion of lipid-based formulations within the intestinal lumen.
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http://dx.doi.org/10.1021/acs.molpharmaceut.6b00887DOI Listing
March 2017

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

J Biomol NMR 2016 11 24;66(3):195-208. Epub 2016 Oct 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met CH assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-C,N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.
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http://dx.doi.org/10.1007/s10858-016-0067-4DOI Listing
November 2016

Structure-Activity Studies of β-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1-RON2 Interaction.

J Mol Biol 2016 10 14;428(20):3986-3998. Epub 2016 Jul 14.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing K values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1-RON2 interaction.
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http://dx.doi.org/10.1016/j.jmb.2016.07.001DOI Listing
October 2016

Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein-Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase.

J Med Chem 2016 06 8;59(12):5799-809. Epub 2016 Jun 8.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria 3052, Australia.

SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II β-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00386DOI Listing
June 2016

Redox-stable cyclic peptide inhibitors of the SPSB2-iNOS interaction.

FEBS Lett 2016 Mar 6;590(6):696-704. Epub 2016 Mar 6.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia.

SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.
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http://dx.doi.org/10.1002/1873-3468.12115DOI Listing
March 2016

Ligand Binding Pathways of Clozapine and Haloperidol in the Dopamine D2 and D3 Receptors.

J Chem Inf Model 2016 Feb 2;56(2):308-21. Epub 2016 Feb 2.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University , 381 Royal Pde, Parkville, Victoria 3052, Australia.

The binding of a small molecule ligand to its protein target is most often characterized by binding affinity and is typically viewed as an on/off switch. The more complex reality is that binding involves the ligand passing through a series of intermediate states between the solution phase and the fully bound pose. We have performed a set of 29 unbiased molecular dynamics simulations to model the binding pathways of the dopamine receptor antagonists clozapine and haloperidol binding to the D2 and D3 dopamine receptors. Through these simulations we have captured the binding pathways of clozapine and haloperidol from the extracellular vestibule to the orthosteric binding site and thereby, we also predict the bound pose of each ligand. These are the first long time scale simulations of haloperidol or clozapine binding to dopamine receptors. From these simulations, we have identified several important stages in the binding pathway, including the involvement of Tyr7.35 in a "handover" mechanism that transfers the ligand between the extracellular vestibule and Asp3.32. We have also performed interaction and cluster analyses to determine differences in binding pathways between the D2 and D3 receptors and identified metastable states that may be of use in drug design.
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http://dx.doi.org/10.1021/acs.jcim.5b00457DOI Listing
February 2016

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

Curr Top Med Chem 2016 ;16(10):1135-53

Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, VIC, 3004, Australia.

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.
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http://dx.doi.org/10.2174/1568026615666150901114329DOI Listing
November 2016

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening.

Proc Natl Acad Sci U S A 2015 Jun 18;112(22):6979-84. Epub 2015 May 18.

Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia; Department of Microbiology, Monash University, Clayton, VIC 3168, Australia; Department of Infectious Diseases, Monash University, Melbourne, 3004, Australia; Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC 3010, Australia

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.
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http://dx.doi.org/10.1073/pnas.1423900112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460473PMC
June 2015